The present invention relates, in general, to compositions and methods aimed at effectively treating anaerobic tumor cells with inhibitors of glycolysis. It also extends to novel and useful methods and compositions for treating aerobic tumor cells with inhibitors of oxidative phosphorylation in combination with glycolytic inhibitors. Inhibiting oxidative phosphorylation to convert aerobic tumor cells to anaerobic cells, hypersensitizes them to glycolytic inhibitors.
U.S. patents that are relevant to the compositions and methods of the present invention are: U.S. Pat. Nos. 4,840,939 and 4,684,627, both for a treatment of cancer with phlorizin and its derivatives; U.S. Pat. No. 4,683,222 for N-glycosylated carboxamide derivatives and their use for influencing the body""s inherent defenses; and U.S. Pat. No. 4,420,489 for sugars with sulfur replacing the ring oxygen atom as antiradiation agents. U.S. Pat. No. 4,840,939 is also relevant for its discussion of the work of Warburg in 1931, concerning the way cancer cells metabolize glucose.
Cancer cells at the inner core of a tumor are poorly oxygenated and consequently rely on anaerobic metabolism for survival. In this condition tumor cells divide more slowly than outer growing aerobic cells and consequently are more resistant to standard chemotherapeutic agents which target rapidly dividing cells. Thus, cells growing anaerobically in these instances exhibit a form of multidrug resistance (MDR) which contributes to chemotherapy failures in the treatment of solid tumors. Anaerobiosis, however, also provides a natural window of selectivity for agents that interfere with glycolysis.
This realization forms the basis for the present invention. According to the present invention, new opportunities are provided for increasing the efficacy of chemotherapeutic protocols. With data and knowledge accumulated by us in our previous work on mitochondrial agents, and with additional work performed by us now, various hypotheses have been formulated and verified to prove the efficacy of the present invention with regard to its compositions and its methods.
See, for example, a series of papers coauthored by one of the present inventors: Lampidis, T J, Bernal, S D, Summerhayes, I C, and Chen, L B. xe2x80x9cRhodamine 123 is selectively toxic and preferentially retained in carcinoma cells in vitro.xe2x80x9d NY Acad. Sci. 397:299-302, 1982; Summerhayes, I C, Lampidis, T J, Bernal, S D, Shepherd, E L, and Chen, L B. xe2x80x9cUnusual retention of Rhodamine 123 by mitochondria in muscle and carcinoma cells.xe2x80x9d Proc. Natl. Acad. Sci. USA 79:5292-5296, 1982; Bernal, S B, Lampidis, T J, Summerhayes, I C, and Chen, L B. xe2x80x9cRhodamine 123 selectively reduces clonogenic ability of carcinoma cells in vitro.xe2x80x9d Science. 218:1117-1119, 1982; Lampidis, T J, Bernal, S D, Summerhayes, I C, and Chen, L B. xe2x80x9cSelective toxicity of Rhodamine 123 in carcinoma cells in vitro.xe2x80x9d Cancer Res. 43:716-720, 1983; and Bernal, S B, Lampidis, T J, McIsaac, B, and Chen, L B. xe2x80x9cAnticarcinoma activity in vivo of Rhodamine 123, a mitochondrial-specific dye.xe2x80x9d Science. 222:169-172, 1983; which showed that Rhodamine 123 (Rho 123) which localizes in mitochondria of living cells, and uncouples ATP synthesis from electron transport, preferentially accumulates in, and kills, a variety of tumor cells as compared to a number of normal cells. We reasoned for the present invention that tumor cells treated with this drug would have to rely solely on glycolysis for ATP production and thus become hypersensitized to inhibitors of glycolysis, like 2-dg (2-dg). In contrast, mitochondrial function in normal cells remained unaffected when treated with Rho 123 and therefore these cells were not hypersensitive to 2-dg. In fact, we found that co-treating human breast carcinoma cells, MCF-7, with Rho 123 and 2-dg, at a dose of Rho 123 that alone inhibited 50% of colony forming units, and at a dose of 2-dg which produced no toxicity, 100% of the colony forming units was inhibited.
This concept was carried over to in vivo studies in which it was found that animals with implanted tumors that were treated in combination with 2-dg and Rho 123 were cured whereas when treated with either drug alone, only partial or no responses were obtained. This latter result provides evidence that manipulation of Oxphos and glycolysis simultaneously can cure tumors in animals. Furthermore, this in vivo data also demonstrates that 2-deoxyglocose can be administered safely to animals, at doses which are effective for anti-tumor activity in combination with an oxphos inhibitor. In this regard, several reports have shown that low levels of 2-dg can be safely administered to animals for various reasons including hypersensitization of tumors to irradiation.
The present invention is based on the discovery that glycolytic inhibitors and analogs thereof, are selectively toxic to tumor cells which are metabolizing anaerobically. Thus, in conjunction with standard chemotherapy and/or radiation which is focused on aerobic, fast-growing cells, which we will here refer to collectively and individually as xe2x80x9caerobic treatmentxe2x80x9d, use of these inhibitors will add to the efficacy of cancer treatment by selectively killing the anaerobically slow-growing tumor cells found at the inner core of solid tumors which are usually the most resistant and consequently the most difficult to eradicate using aerobic treatments.
As an aid for explaining and for demonstrating the usefulness of the present invention, we refer to the equation (A)=(B)=(C) where:
(A)=tumor cells treated at a dose of rhodamine 123 which specifically uncouples ATP synthesis from electron transport;
(B)=xcfx81o cells which are cells that contain no mitochondrial DNA and therefore cannot undergo oxidative phosphorylation; and
(C)=tumor cells which are growing anaerobically.
In all three classes, the cells can only produce ATP via the Embden-Myerhoff pathway (glycolysis) and thus are naturally hypersensitive to all inhibitors of glycolysis exemplified by 2-deoxyglucose, oxamate and novel compounds of the present invention.
Although Warburg originally proposed that tumor cells depend less on mitochondrial function for ATP production and more on glycolysis than normal cells, there has been no definitive data to date to confirm this hypothesis. There is data however which indicates that cells, tumor or normal, which are compromised aerobically and switch to anaerobic metabolism increase their uptake and utilization of glucose. The increased uptake has been attributed to the increased appearance of glucose receptors on the plasma membrane. The fact that cells growing at the inner core of tumors rely more on glycolysis than cells on the outer edge most likely accounts for the successful use of the radioactive analog of glucose, 2-deoxyglucose (2-dg), as a diagnostic tool for localizing tumors. This has been the main usage of 2-dg for cancer. Moreover, it has been suggested that 2-dg is taken up more by anaerobic cells due to enhanced expression of glucose receptors. Thus, since a cell that is functioning normally relies mainly on oxidative phosphorylation for its supply of ATP when this mechanism is compromised or absent, glycolysis (the only other way of producing ATP) automatically becomes enhanced. This is precisely why inner core tumor cells should naturally become hypersensitive to agents which block glycolysis, i.e. 2-dg and others that block the glycolytic pathway at different steps, such as oxamate and iodoacetate.
Oxamate is another agent which blocks glycolysis and is more specific than 2-dg for anaerobically metabolizing cells. Oxamate has been shown to inhibit lactic dehydrogenase, the enzyme which breaks down pyruvate to lactic acid. Thus, the fact that oxamate blocks glycolysis at a different step than 2-dg, and is clearly active in our in vitro cell systems in selectively killing anaerobic cells (models A and B), lends further proof that our discovery is correct and works according to the principles we set forth in this application. The enzyme reaction which converts pyruvate to lactate occurs only when the cell""s ability to process oxygen becomes limited. Thus, aerobically metabolizing cells do not use this enzyme and inhibitors of this reaction will inherently be more selective than inhibitors of glycolysis such as 2-deoxyglucose since these latter agents inhibit reactions that occur in aerobic as well as anaerobic cells.
Iodoacetate, on the other hand, is known to inhibit glycolysis at another step in the pathway, namely at glyceraldehyde 3-phosphate dehydrogenase, which normally yields 1,3 biphosphoglycerate from glyceraldehyde 3-phosphate and requires NAD+ to proceed.
The inventors have investigated this overall concept by utilizing an osteosarcoma cell line that has been selected for loss of mtDNA, and thus cannot produce ATP by oxidative phosphorylation (cells (B) above). The inventors have also utilized this concept as a model to determine whether any test drug utilizes functional mitochondria as a cytotoxic target. The model indicates that a drug which interferes with ATP production in mitochondria of whole cells, but not necessarily in isolated mitochondrial preparations, will hypersensitive a cell to glycolytic inhibitors, i.e. 2-dg, oxamate and iodoacetate. Data has been developed by the inventors which clearly shows that in the cases listed as (A) and (B) above, both are hypersensitive to 2-deoxyglucose, oxamate and iodoacetate which forms the basis for the present invention.
The data which supports the invention that anaerobic cells within a tumor are selectively sensitive to glycolytic inhibitors, indicates that this is a universal phenomenon and that most or all glycolytic inhibitors will, in general, be similarly effective.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and specific objects attained by its uses, reference is made to the accompanying descriptive matter in which preferred embodiments of the invention are illustrated.